The present invention relates to 4-alkyl substituted 3,4-dihydropyrrolo[1,2-a]pyrazin-1(2H)-one derivatives, to a process for their preparation, to the pharmaceutical compositions comprising them, and to their use as therapeutic agents, particularly in the treatment of diseases caused by dysregulated protein kinase activity, such as cancer, cell proliferative disorders, viral infections, immune disorders, neurodegenerative disorders and cardiovascular diseases. The malfunctioning of protein kinases (PKs) is the hallmark of numerous diseases. A large share of the oncogenes and proto-oncogenes involved in human cancers encodes for PKs. The enhanced activities of PKs are also implicated in many non-malignant diseases, such as benign prostate hyperplasia, familial adenomatosis, polyposis, neurofibromatosis, psoriasis, vascular smooth cell proliferation associated with atherosclerosis, pulmonary fibrosis, arthritis glomerulonephritis and post-surgical stenosis and restenosis.
PKs are also implicated in inflammatory conditions and in the multiplication of viruses and parasites. PKs may also play a major role in the pathogenesis and development of neurodegenerative disorders.
For a general reference to PKs malfunctioning or deregulation see, for instance, Current Opinion in Chemical Biology 1999, 3, 459-465; Nature Rev. Drug Discov. 2002; and Carcinogenesis 2008, 29, 1087-1091.
Originally identified as activated genes by proviral mutagenesis in a lymphoma mouse model, PIMs (PIM1, PIM2 and/or PIM3 throughout this application) are protein-serine/threonine kinases. PIM kinases are poorly expressed in normal tissues, and overexpressed or even mutated in a discrete number of human cancers, including Lymphoma, Leukaemia, Prostate, Pancreas and Gastric cancers [Shah et al. Eur. J. Cancer, 44, 2144-51, (2008)].
PIM kinases are constitutively active and their activity supports in vitro and in vivo tumor cell growth and survival through modification of an increasing number of common as well as isoform-specific substrates, including several cell cycle regulators and apoptosis mediators. PIM1 but not PIM2 seems also to mediate homing and migration of normal and malignant hematopoietic cells by regulating chemokine receptor surface expression [Brault et al. Haematologica 951004-1015 (2010)].
There is an increasing evidence that PIM1 and PIM2 kinases may be involved in mediating the oncogenic effects of some acute myelogenous leukemias (AML)-associated oncogenes, in particular, the oncogenic role of FLT3-mutations (ITD and KD mut., present in 30% of AMLs) and/or translocations involving the MLL gene (occurring in 20% of AMLs), [Kumar, et al. J. Mol. Biol. 348, 183-193, (2005)]. PIM1 is more expressed in FLT3-ITD-transformed AML cells than in WT bone marrow cells. Data suggest that PIM1 as well as PIM2 inhibition may mediate FLT3-ITD-dependent death of AML cells. Interestingly, cells transformed by FLT3 mutations that confer resistance to small-molecule tyrosine kinase inhibitors were still sensitive to knockdown of PIM2, or PIM1 and PIM2 by RNAi, [Kim et al., Blood 105, 1759-67, (2005)].
Moreover, PIM2 has been reported being over-expressed and associated with progression of several malignancies that originate from the B-cell lineage such as chronic lymphocytic (CLL), diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma (MCL) or myeloma [Cohen et al., Leukemia & Lymphoma 45(5) 951-955 (2004), Huttmann et al. Leukemia 20 1774 (2006)].
In recent studies, it was demonstrated that both NF-κB and Pim kinases are implicated in tumorigenesis, in particular, PIM1 phosphorylation of RelA/p65 at Ser276 is believed to allow defense against ubiquitin-mediated degradation and whereby exerted activation of NF-κB signalling, [Nihira K. et al. Cell Death & Differentiation 2010, 17, 689-698].
In prostate cancers, oncogenic PIM1 kinase is implicated with c-Myc in carcinogenesis, and the c-MYC/Pim1 synergy is critically dependent on PIM1 kinase activity. PIM1 cooperativity with c-MYC in vivo, is explained not only on the c-MYC activity by S62 phosphorylation, but also on the evidence of neuroendocrine (NE) differentiation [Wang J. et al. Oncogene (2010) 29, 2477-2487].
Interestingly, PIM and AKT/PKB seem to play partly redundant roles in mediating growth and survival of hematopoietic cells most probably due to overlapping substrates like BAD, p21WAF1/CIP1, p27KIP1, or Cot/Tpl-2 [Choudhary et al., Mol. Cell. 36 326-39 (2009)].
PIM kinases have been shown to control mTOR inhibition (rapamycin) resistance, proliferation and survival. Therefore, a combination of small molecule inhibitors targeting several survival kinases might be essential for a powerful cancer therapeutic platform [Amaravadi R., et al. J. Clin. Invest. 2005, 115 (10) 2618-24]. Oncogenic protein synthesis through eIF4E binding protein 1 (4E-BP1) seems to be mTOR-independent and controlled by PIM2. These observations suggest that the oncogenic eIF4F translation-initiating complex could be blocked with small molecules PIM2 inhibitors [Tamburini J. et al. Blood 2009, 114 (8), 1618-27; Brault L. et al. Haematologica 2010, 95 (6) 1004-1015 and Beharry Z. PNAS 2011 108, 528-533].
Recently two different research groups have reported the successful combination of PIM and PI3K inhibitors. Blanco-Aparicio, C. et al. [Cancer Lett. 2011, 300(2):145-53] combined the PI3K inhibitor GDC-0941 with a PIM1 inhibitor and found a strongly synergistic effect in AML cells. Ebens et al. during the 52nd ASH annual meeting, reported that a pan-PIM inhibition suppressed growth in myeloma cell lines, xenografts, and primary patient samples, both as a single-agent as well as acting synergistically in combination with GDC-0941.
3,4-Dihydro-2h-pyrrolo[1,2-a]pyrazin-1-one derivatives possessing kinase inhibitory activity have been disclosed in WO2010/031816, in the name of the Applicant itself.